Project Summary/Abstract: Ventricular Arrhythmia is a leading cause of death following Myocardial Infarction (MI), with high incidence among aging populations. Recent data from our laboratory show that the osmotically activated Transient Receptor Potential Vanilloid 4 (TRPV4) cation channel increases expression in ventricular cardiomyocytes with advancing age. Ischemia-Reperfusion (I/R) is associated with both Ca2+ overload and hypoosmotic stress on the myocardium. Ca2+ overload disturbs excitation-contraction coupling and promotes arrhythmia by increasing diastolic Ca2+ release and membrane depolarization. Thus, this proposal tests the hypothesis that hypoosmotic stress during I/R activates TRPV4-mediated Ca2+ influx, which contributes to Ca2+ overload and arrhythmogenesis. Specific Aim 1 is to investigate the role of TRPV4 in isolated cardiomyocyte Ca2+ influx, membrane potential, and Ca2+ homeostasis following hypoosmotic stress. Patch clamp recording of membrane potential and membrane Ca2+ currents will be measured in cardiomyocytes from Aged mice (24-26 month, with high TRPV4 expression), Young mice (3-6 month, with low TRPV4 expression), and Young mice with inducible transgenic cardiac-specific TRPV4 overexpression (Young TRPV4 Overexpressors). Isolated cardiomyocytes from the same groups, but with an additional GCaMP6f Ca2+ sensor transgene??will also be subjected to hypoosmotic stress and intracellular Ca2+ homeostasis will be assessed using laser scanning confocal microscopy in the presence and absence of TRPV4 antagonism (HC067047, 1 M). Specific Aim 2 is to investigate the arrhythmogenic role of TRPV4 in Langendorff-perfused isolated hearts subjected to I/R. Reversible ligation of the left anterior descending artery (LAD) will induce I/R in Aged, Young, and Young TRPV4 Overexpressor hearts. Sharp microelectrode recordings of cardiomyocytes from LAD-supplied myocardium will be taken prior to, during, and following I/R in the presence and absence of TRPV4 antagonism. The same protocol will be conducted on Langendorff perfused Young and Young TRPV4 Overexpressor isolated hearts (with the GCaMP6f Ca2+ sensor) while imaging of the left-ventricular free wall by laser scanning confocal microscopy. This in situ imaging will allow for measurement of arrhythmogenic diastolic Ca2+ release events in the intact heart during I/R. This project investigates a novel therapeutic target for preventing Ca2+-dependent ventricular arrhythmias following myocardial infarction in aging populations. Additionally, the proposal provides valuable training to the applicant in conducting cardiovascular research.